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US 12,435,355 B2Granted
Whole transcriptome analysis in single cells
AssigneeRoche Sequencing Solutions, Inc.
InventorsSedide Ozturk, Martin Ranik, Florian Rubelt
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Abstract
The invention is a method of single cell transcriptome analysis. The method comprises detecting multiple transcripts in each individual cell of the plurality of cells by barcoding the transcripts with a cell-specific compound barcode formed using a DNA polymerase and a terminal transferase, optionally in a single enzyme such as a reverse transcriptase.
Claims20
A method of detecting a plurality of target nucleic acids in a plurality of cells in a sample, the method comprising:
- (a) contacting one or more target nucleic acids in each cell of the plurality of cells in the sample with an oligonucleotide primer, wherein the contacting is performed in the presence of a nucleic acid polymerase having terminal transferase activity and template switch activity;
- (b) for each of the one or more target nucleic acids in each cell of the plurality of cells in the sample, forming a copy strand by extending the oligonucleotide primer with the nucleic acid polymerase, wherein the copy strand includes a 3′-end having one or more non-templated nucleotides, and wherein the one or more non-templated nucleotides at the 3′-end of the copy strand serve as a site for annealing a barcode subunit;
- (c) generating a cell-characteristic compound barcode for the one or more target nucleic acids in each cell of the plurality of cells in the sample, wherein the one or more target nucleic acids in different cells of the plurality of cells include different cell-characteristic compound barcodes, while the one or more target nucleic acids within any one individual cell of the plurality of cells include the same cell-characteristic compound barcode, wherein each cell-characteristic compound barcode comprises concatemerized barcode subunits, and wherein the generating of the cell-characteristic compound barcodes in each cell of the plurality of cells in the sample comprises repeatedly performing split-pool synthesis rounds until the one or more target nucleic acids in each of the different cells of the plurality of cells include the different cell-characteristic compound barcodes, wherein each split-pool synthesis round comprises:
- (i) randomly splitting the plurality of cells in the sample into a plurality of reaction volumes, wherein each reaction volume of the plurality of reaction volumes comprises a unique barcode subunit and the nucleic acid polymerase, wherein each unique barcode subunit comprises a barcode and a nucleic acid sequence complementary to the one or more nucleotides at the 3′-end of the copy strand;
- (ii) extending the 3′-end of the copy strand with the nucleic acid polymerase, wherein the extending of the 3′-end of the copy strand copies the unique barcode subunit and adds a strand of non-templated nucleotides to the 3′-end of the copy strand;
- and
- (iii) combining the plurality of reaction volumes;
- and
- (d) determining the sequence of the resulting extended copy strand for each of the one or more target nucleic acids in each cell of the plurality of cells including the generated cell-characteristic compound barcodes, thereby detecting each target nucleic acid of the plurality of target nucleic acids in the plurality of cells.
6 more elements
A method of detecting a plurality of target nucleic acids in a plurality of cells in a sample, the method comprising:
- (a) hybridizing an oligonucleotide primer to one or more target nucleic acids in each cell of the plurality of cells in the sample;
- (b) for each of the one or more target nucleic acids in each cell of the plurality of cells in the sample, forming a copy strand by extending the hybridized oligonucleotide primer with a nucleic acid polymerase having terminal transferase activity and template switch activity, wherein the copy strand includes a 3′-end having one or more non-templated nucleotides, and wherein the one or more non-templated nucleotides at the 3′-end of the copy strand serve as a site for annealing a barcode subunit;
- (c) generating a cell-characteristic compound barcode for the one or more target nucleic acids in each cell of the plurality of cells in the sample, wherein the one or more target nucleic acids in different cells of the plurality of cells include different cell-characteristic compound barcodes, while the one or more target nucleic acids within any one individual cell of the plurality of cells include the same cell-characteristic compound barcode, wherein each cell-characteristic compound barcode comprises concatemerized barcode subunits, and wherein the generating of the cell-characteristic compound barcodes in each cell of the plurality of cells in the sample comprises repeatedly performing split-pool synthesis rounds until the one or more target nucleic acids in each of the different cells of the plurality of cells include the different cell-characteristic compound barcodes, wherein each split-pool synthesis round comprises:
- (i) randomly splitting the plurality of cells in the sample into a plurality of reaction volumes, wherein each reaction volume of the plurality of reaction volumes comprises a unique barcode subunit and the nucleic acid polymerase, wherein each unique barcode subunit comprises a barcode and a nucleic acid sequence complementary to the one or more nucleotides at the 3′-end of the copy strand;
- (ii) extending the 3′-end of the copy strand with the nucleic acid polymerase, wherein the extending of the 3′-end of the copy strand copies the unique barcode subunit and adds a strand of non-templated nucleotides to the 3′-end of the copy strand;
- and
- (iii) combining the plurality of reaction volumes;
- and
- (d) determining the sequence of the resulting extended copy strand for each of the one or more target nucleic acids in each cell of the plurality of cells including the generated cell-characteristic compound barcodes, thereby detecting each target nucleic acid of the plurality of target nucleic acids in the plurality of cells.
6 more elements
Dependencies are inferred from the claim text.